Macrolides have long been among the most widely used antibiotics. Despite this utility, development of new macrolides through traditional synthetic and semisynthetic approaches has been greatly hindered by the inherent structural complexity of these compounds. Precursor-directed biosynthesis is a technique which circumvents this difficulty by incorporating simple synthetic precursors into a biosynthetic pathway, allowing the bulk of the molecule to be constructed enzymatically. This dissertation describes the evolution and application of a system for the facile production of new macrolides through precursor-directed biosynthesis. The results of this work are the discovery of an unexpected macrolide structure-activity relationship and the ultimate discovery of a promising new lead for macrolide development.